Project's objective
Our objective is to use plants as CO2 sensors. In order to achieve this, we had to search for inducible promoters to high concentrations of CO2. We have then tested them by constructing a plasmid with the Gateway Cloning Method
Design
Our constructions had the following parts: a high CO2 inducible promoter, the GPF marker gene, and a terminator. To test the selected promoters, we created 11 constructions with this structure using the Gateway cloning method.
Once we had the constructions we electroporated them in Agrobacterium tumefaciens to infiltrate Nicotiana. When the plants were transformed we exposed them to high levels of CO2 to compare GFP expression with transformed plants at ambient CO2.
Cloning method
Gateway is a clonation method developed by Invitrogen and is the method we have used to construct our plasmids. Briefly explaining, the gateway method is divided into two steps.
In the first step, we introduce our selected promoter in the pDONR plasmid by the recombination of the attB1/attB2 and attP1/attP sites. This way, the promoter substitutes the ccdB gene, a lethal gene that inhibits helicases and as a result, we have our pDONR plasmid with our promoter.
In the next step, we transfer our promoter into the final plasmid which already has the GFP and the terminator. In this second reaction, the recombination is between attL1/attL2 and attR1/attR2 sites to substitute ccdB of the final plasmid by our promoter. As a result, we have our complete transcriptional unit, promoter, GFP, and terminator in the final plasmid (see “Parts”).
Practical demostration
The infiltration process consists of introducing transformed Agrobacterium tumefaciens, a bacteria that naturally infects wounded plants and transfers part of its DNA to the plant cell, into our Nicotiana benthamiana with a syringe. The bacteria are introduced between the two layers of the epidermis of the leaves. For the process to be efficient we had to infiltrate in leaves that were between 3 and 5 cm in diameter, which normally corresponds to young plants with tender leaves.
After a few days we divided our transformed plants in two groups: one was exposed to high concentrations of CO2 (1,000 ppm) for 24 h and the other one was left at ambient CO2 levels (400 ppm) as control.
Finally we analyzed the samples on a confocal microscope, to see GFP expression(See “Protocols” section to see all the explanations of the process).
Our goal was to identify plants that expressed more GFP under high CO2 conditions than under control conditions. Throughout the entire project, we have been improving different steps, such as the time between the moment of infiltration and the GFP analysis, plant growth conditions, time CO2 exposure…
Success
From the eleven tested promoters, we have encountered significant differences between exposed and control plants in three cases. These promoters are: Citrate synthase 4 (CSY4), Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) and Adenosine Triosephospate Synthase (ATPS). For more data go to the “Results” section.
